Method for diagnosing Alzheimer disease

ABSTRACT

The invention concerns a method for diagnosing Alzheimer disease, consisting in demonstrating one or several mutations in the genomic DNA region regulating the expression of the apolipoprotein E gene, inducing a modification of the apolipoprotein E gene, with respect to a control population or a modification of the expression relative to the alleles of the apolipoprotein E gene.

The present invention relates to a method for diagnosing Alzheimer'sdisease.

Alzheimer's disease is a neurodegenerative dementia characterized by aloss of cortical neurons associated with β-amyloid plaques, ofneurofibrillary tangles and, in most cases, an amyloid angiopathy. It isstrongly suspected that there is a genetic influence in the aetiology ofAlzheimer's disease (WO 94/01772).

This genetic component has been brought to the fore over many years byindirect observations which suggest that the disease is inherited in anautosomal dominant fashion with an age-dependent penetrance in order toexplain certain familial forms of Alzheimer's disease. Recent moleculargenetic studies have enabled putative genes for Alzheimer's disease tobe isolated by looking for chromosome-specific polymorphic geneticmarkers (Bird et al., 1989, Neurobiology of Aging 10, 432-434).

Four chromosomal localizations have been described as being involved:three on chromosomes 1, 14 and 21 in the early onset familial forms (ageat onset under 60 years), and one on chromosome 19 in the late onsetfamilial and sporadic forms. Two linkage studies have suggested that thechromosomal region 19q13.2 was associated with late onset familial formsof Alzheimer's disease (Pericak-Vance et al, Am. J. Hum. Genet. (1991),48, 1034-1050). Within this chromosomal region, the group of genes forapolipoproteins (APO) E-CI-CI′-CII is a candidate zone. Among theproducts of these genes, apolipoprotein E (APOE) is involved especiallyin the nervous system APOE is present in the senile plaques andpossesses a binding affinity for the peptide Aβ. APOE is characterizedby three major alleles ε2, ε3, ε4. Strittmatter et al. (Proc. Natl.Acad. Sci. (1993) 90, 177-181) have described an increased frequency ofthe ε4 allele of the APOE gene in the late onset familial forms ofAlzheimer's disease. This observation has been confirmed for thefamilial forms (Corder et al., Science (1993), 261, 921-923) and thesporadic forms of Alzheimer's disease (Corder et al., Science (1993),261, 921-923; Saunders et al., Neurology (1993), 13, 1467-1472).

FR-2 716 894 describes a method which makes it possible toprognosticate, for a given disease, the risks of developing Alzheimer'sdisease with respect to the general population. This method is based onthe detection of the ε4 alleles of the APOE gene, of the short allelesof the marker D19S178 and of the long alleles of the APO CII gene, alllocalized on chromosome 19.

Several hypotheses make it possible to explain this phenomenon.

Recent studies by the inventors, who are the authors of the presentinvention, now confirm the hypothesis of the existence of at least oneother functional mutation in the 19q13.2 region.

Indeed, in addition to a functional effect specific to the polymorphismof apolipoprotein E, the studies by the inventors show differences inlevels of expression which are significant in sick subjects comparedwith healthy controls, which indicates that one or more mutations in theregulatory regions of the APOE gene are involved in the onset ofAlzheimer's disease. Furthermore, relative differences in expression arefound in the controls.

The inventors have more particularly identified a new polymorphism inthe region of the promoter of the gene encoding the apolipoprotein Eprotein found in a potential binding site for the Th1/E47cstranscription factors.

For the determination of this polymorphism, the sequence described byPaik et al. (1985, Proc. Natl. Aca. Sci., vol. 82, p. 3447) will betaken as reference.

This polymorphism has been called by the inventors Th1/E47cs forTh1/E47cs consensus, since it is situated in a consensus sequence forbinding of the Th1/E47cs transcription factor.

The mutation identified is characterized by a Thymine to Guaninesubstitution (G→T) in the sequence:

GGGTGTCTGT(or G)ATTACTGGG,

G being the most frequent allele in the normal population.

The alleles corresponding to this polymorphism are called hereinafter T(when the base is Thymine) or G (when the base is Guanine).

The determination of the allele can be carried out after PCRamplification of the DNA region comprising this polymorphism:

either by creating in one of the PCR primers a cleavage site for arestriction enzyme not existing in individuals carrying one of thealleles,

or by a hybridization technique using oligonucleotide probes specificfor the alleles.

The inventors have studied the influence of the Th1/E47cs polymorphismon the expression of the alleles of the APOE gene and demonstrated anincrease in the risk of developing Alzheimer's disease associated withthe T allele of Th1/E47cs, specific to this allele and not due to the ε4allele.

Furthermore, the Th1/E47cs polymorphism modulates the risk associatedwith the ε4 allele in individuals with the ε3/ε4 genotype, theindividuals with the GT genotype having an increased risk of developingAlzheimer's disease compared with the individuals homozygous for theTh1/E47cs polymorphism. Reinforcing this observation, the authors haveconsidered that the combination of the T allele of Th1/E47cs with the ε4allele on the same chromosome corresponds to the most unfavourablecombination.

The subject of the invention is thus a method for diagnosing Alzheimer'sdisease, comprising the identification of one or more mutations in thegenomic DNA region for regulating the expression of the apolipoprotein Egene, inducing a modification of the expression of the apolipoprotein Egene relative to a control population or a modification of the relativeexpression of the alleles of the apolipoprotein E gene.

For the purposes of the present invention, diagnosis is understood tomean the confirmation of a mutation in the regulatory region of the APOEgene in a patient whose clinical picture signals a symptomatology whichmay be attributed to Alzheimer's disease, or alternatively an increasedprobability in subjects of developing Alzheimer's disease relative tothe population as a whole, the increase in probability beingstatistically significant.

The chromosomal DNA region for regulating the APOE gene is broadlydefined as being the chromosomal region 19q13.2 other than the regionencoding apolipoprotein E (Human Molecular Genetics, 1994, vol. 3, No.4, 569-574).

Advantageously, the chromosomal DNA region in which one or moremutations are identified is situated between the marker D19S178 and theAPOCII gene, and comprises more particularly the introns and theflanking regions of the APOE gene, extending over a distance of 5 kbupstream and downstream of the APOE gene.

The subject of the invention is more particularly a method fordiagnosing Alzheimer's disease comprising the identification of at leastone mutation in the promoter of the APOE gene, situated at 186 basesfrom the TATA box of this gene, the mutation consisting moreparticularly of the replacement T→G in the sequence defined above.

More particularly, the method consists in testing for one or moremutations in the region of the promoter of the gene encodingapolipoprotein E, existing in particular in a potential binding site forthe Th1/E47 transcription factors.

The subject of the invention is also a method for diagnosing Alzheimer'sdisease comprising a determination of the genotype of apolipoprotein Eand the test for a mutation of the type described above in theregulatory region of the APOE gene.

In accordance with this diagnostic method, the presence of at least oneε4 allele of apolipoprotein E conjointly with the existence of amutation in the regulatory region of the APOE gene, in particular themutation defined above inducing a modification of the expression of theAPOE gene or a relative difference in expression of the alleles ofapolipoprotein E where appropriate, will range towards the diagnosis ofAlzheimer's disease in patients whose clinical picture presents asymptomatology which can be attributed to Alzheimer's disease or willmake it possible to classify subjects in good health in a category withan increased risk of developing Alzheimer's disease.

Relative difference in expression of the alleles is understood to mean adifference in expression of one allele relative to another,independently of the absolute level of expression of the gene.

The test for the ε4 allele is done by any appropriate method based onthe presence of an ARG residue at position 112 of apolipoprotein E forthe ε4 allele, of a CYS residue at position 158 for the ε2 allele,relative to the residues CYS and ARG in these positions for the ε3isoform, which is the most widespread.

The identification of an additional mutation in the regulatory regionsof the APOE gene as defined above is carried out by any appropriatemethod, in particular a method for diagnosing Alzheimer's diseasecomprising the identification of at least one mutation in the promoterof the gene encoding APOE, situated at 186 bases from the TATA box ofthis gene.

The presence of at least one ε4 allele of the APOE gene, of at least oneshort allele of the marker D19S178 and of at least one long allele ofthe APO CII gene as described in FR-2,716,894 and the existence of atleast one mutation in the regulatory region of the APOE gene willstrongly contribute towards orienting the diagnosis of Alzheimer'sdisease in symptomatic subjects or will constitute a substantial riskfactor in asymptomatic subjects.

Since the mutation(s) involved in Alzheimer's disease are responsiblefor a variation in the relative expression of the alleles of the APOEgene in the brain, it is also possible to determine, instead of or inaddition to a mutation in the regulatory regions of the APOE gene whichare defined above, the level of expression of the APOE gene and tocompare this level of expression to that of the general population.

The diagnostic method based on the determination of the level ofexpression of apolipoprotein E is particularly advantageous inheterozygous subjects, in particular carrying the ε4 allele. In thesesubjects, the diagnostic method for the purposes of the inventionadvantageously comprises the determination of the level of expression ofthe ε4 allele relative to the ε2 or ε3 allele.

In the same manner, the diagnostic method in the individuals with theε2/ε3 genotype advantageously comprises the determination of the levelof expression of the ε2 allele relative to the ε3 allele.

An increase or a significant decrease, respectively, in the level ofexpression/transcription of the ε4 allele in a heterozygous subject ε4ε2or ε4ε3 and of the ε2 allele in a heterozygous subject ε2ε3, will orientthe diagnosis towards a dementia of the Alzheimer type, if moreover thesubject presents a clinical picture evoking the symptomatology ofAlzheimer's disease. In a subject not presenting apparent clinicalsigns, the increase or the decrease in expression of the ε4 and ε2alleles, respectively, will be an indication of an increased probabilityin the subject of subsequently developing Alzheimer's disease.

The determination of the level of expression of the APOE gene and moreparticularly of the ε4 and ε2 alleles of the APOE gene is advantageouslycarried out by measuring the relative level of the mRNA for the APOEgene either by establishing the ratio of transcription of the ε4 allelerelative to the ε2 or ε3 allele in the case of individuals with the ε4ε2and ε4ε3 genotypes, or by establishing the ratio of transcription of theε2 allele relative to the ε3 allele in the case of individuals with theε2ε3 genotype.

The measurement of the level of transcription is carried out followingthe extraction of mRNA from biopsy tissues or from cells in cultures andamplification by RT-PCR (reverse transcription polymeric chainreaction), with the aid of appropriate primers specific for the allelefor which it is desired to measure the level of expression.

The tissue used is for example derived from a biopsy of cerebral tissue,in particular of frontal lobes, thus making it possible to measure thelevel of expression of the alleles of APOE in the brain. It is alsopossible to determine the level of transcription of the mRNAs for APOEin lymphocytes or fibroblasts in cell culture. In general, it will bepossible to determine the level of transcription of the alleles of APOEin any tissue capable of exhibiting a variation in the percentage ofexpression of an allele relative to another between the patients andthose who are sick. Likewise, this method can also be applied for thedevelopment and the use of cellular or animal models using the allelesof APOE.

The ratio of the expression of the alleles of the APOE gene isdetermined in the following manner:

a) the cDNA is subjected to a PCR amplification in the presence ofprimers permitting the specific amplification of at least onepolymorphic sequence of the alleles;

b) the amplified DNA is subjected to the action of at least onerestriction enzyme, permitting the differentiation of the alleles;

c) the DNA fragments are separated;

d) the quantity of fragments obtained is evaluated by means of a markeremitting a detectable signal;

e) the initial ratio in the different alleles is determined by thefollowing formula:$\frac{N_{o\quad {allele1}}}{N_{o\quad {allele1}} + N_{o\quad {allele2}}} = \frac{A\quad \alpha_{allele1}^{\prime}}{{A\quad \alpha_{allele1}^{\prime}} + \alpha_{allele2}^{\prime}}$

in which N_(o) is the initial number of DNA molecules,

A is the coefficient of proportionality which makes it possible tocorrect the size difference between the different restriction fragmentsand is equal to the ratio of the lengths of restriction fragmentscharacteristic of each allele,

and α′ is determined

either by the following formula:$\alpha^{\prime} = \frac{O\quad D_{\max}}{K^{\prime}}$

 in which OD_(max) is the maximum optical density which may be measured,

K′ is a constant,

OD_(max) and K′ being determined by the following function f:${O\quad D} = {{f(V)} = {\frac{O\quad D_{\max}}{\left( {K^{\prime} + V} \right)} \cdot V}}$

 in which V is the volume of the sample obtained by PCR subjected tostep c) and OD is the optical density measured;

or by the following function g:$\frac{1}{Q\quad D} = {{g\left( \frac{1}{V} \right)} = {{\frac{1}{\alpha^{\prime}} \times \frac{1}{V}} + \frac{1}{O\quad D_{\max}}}}$

in which OD is the optical density measured, V is the volume of thesample obtained by PCR subjected to step c) and OD_(max) is the maximumoptical density which may be measured;

the coefficient of amplification E₁ of the DNA containing allele 1 beingidentical to the coefficient of amplification E₂ of the DNA containingallele 2, for the different alleles of APOE.

The DNA amplified in step a) is, according to the invention, a cDNAcomprising the allelic sequences of interest, obtained from mRNA by theusual technique of RT-PCR. The alleles are differentiated according tosteps b) and c) described above, which demonstrate the restrictionpolymorphisms (RFLP).

The separation of the DNA fragments according to step c) may be carriedout in particular by gel electrophoresis, preferably by polyacrylamidegel electrophoresis.

In the case of the APOE gene, the ε3, ε2 and ε4 alleles may becharacterized by restriction fragments of 91 bp, 83 bp and 72 bp,respectively.

The coefficient of proportionality A can therefore be calculated asbeing A=91/72 for the ε3/ε4 alleles, A being 83/72 for the ε2/ε4individuals and A being 91/83 for the ε2/ε3 individuals. For the lattergenotype, since the two ε2 and ε3 alleles give a restriction fragmentlength of 91 bp, the relationshipAOD_(allele 1)+OD_(allele 2)=OD_(91 bp) is produced.

This method is in addition particularly simple. It is indeed sufficienteither to present the value of the optical density (OD) as a function ofthe volume V of the sample, it being possible for the representativecurve of the function f such that OD=f(V) to be plotted using the methodof least squares, and to determine the OD_(max) and K′ from this curve;or to present the value 1/OD as a function of the reciprocal of thevolume of the sample, that is to say 1/V, it being possible for therepresentative curve of the function g such that$\frac{1}{O\quad D} = {g\left( \frac{1}{V} \right)}$

to be plotted by linear regression, the slope of the straight lineobtained being equal to the reciprocal of α′.

The subject of the invention is also a method for diagnosing Alzheimer'sdisease comprising the determinations of the phase of the differentpolymorphisms ε, and Th1/E47cs and optionally of −491 AT and 1E1 whichare capable of bringing about an increased risk of developingAlzheimer's disease.

At the level of the population with the ε3ε4 genotype, the riskassociated with the GT genotype is greater than that associated with theGG genotype. This observation can be explained by taking into accountthe phase of the polymorphisms Th1/E47cs and ε2, ε or ε4 of APOE.Indeed, at the level of the heterozygotes GT and ε3ε4, the risk ofdeveloping the disease depends on the combination of the G allele and ofthe ε4 allele on the same chromosome, thus determining the phase of thepolymorphisms ε and Th1/E47cs of APOE. Following this phase, twopossibilities exist: (i) a higher level of expression of ε4 (ii) ahigher level of expression of the ε3 allele. The first possibility wouldcause a physiological response in favour of the properties of theisomorph APOε4 and would explain a more marked effect at the level ofthe individuals carrying the ε3ε4 and GT genotypes. This difference inthe level of expression linked to the phase will be true for allheterozygous genotypes. However, the risk associated with the G alleleis probably attenuated because of the different combinations which arepossible between the ε and Th1/E47cs polymorphisms of APOE.

In accordance with the invention, the phase of the polymorphisms of APOEand of Th1/E47cs is studied in the following manner:

A fragment of 4600 bp containing the ε polymorphisms of APOE andTh1/E47cs is amplified by PCR. This amplification is carried out usingthe kit: extend long template PCR system (Boehringer) with as senseoligonucleotide;

5′-GGGGGAGGTGCTGGAATCT-3′

and as antisense oligonucleotide:

5′-CAGATGCGTGAAACTTGGTGA-3′.

The product of amplification is then digested with the restrictionenzyme Afl III, whose sole cleavage site on the amplified fragment makesit possible to differentiate the ε4 allele from the ε3 allele. Aftermigration of the product of digestion on a 0.8% agarose gel, the DNA istransferred onto nitrocellulose membrane and fixed under UV. Thediscrimination between the T allele and the G allele is carried out by aprotocol similar to that used for the ASO genotyping of thispolymorphism.

Depending on the phases of the APOE and Th1/E47cs polymorphisms, it willbe possible to define subgroups of sick subjects and to determine foreach subgroup the optimum therapy. Likewise, the determination of thephase with other polymorphisms of the regulatory regions of the APOEgene capable of influencing (or otherwise) its expression, such as −491AT described by Bullido et al., 1E1, APO CI, APO CII and D19S178 couldalso prove useful for the determination of subgroups of subjects at highrisk of developing the disease.

Thus, it is known that subjects suffering from Alzheimer's disease havea predisposition to respond to certain therapies, in particular totherapies of the cholinomimetic type depending on the type and thenumber of copies of the alleles of the APOE gene.

The subject of the invention is therefore also a method for identifyingsubjects suffering from Alzheimer's disease who are capable ofresponding to a given therapy, for example of the cholinomimetic type,comprising:

a) the determination of the genotype of apolipoprotein E;

b) the determination of the genotype of the Th1/E47cs polymorphism; and

c) optionally, the determination of the phase of the polymorphisms ofapolipoprotein E and of Th1/E47cs.

The different polymorphisms of APOE and of Th1/E47cs may be exploited tocreate animal or cellular models expressing APOE better or less well,used alone or in combination with the genetic factors capable ofinfluencing the development of the pathology such as APP, PS1, PS2, andthe like, or the other markers of Alzheimer's disease such as abnormalphosphorylation of the protein Tau.

The subject of the invention is therefore also a transfection vector foreukaryotic cells comprising at least one allele of the APOE gene and oneallele of the Th1/E47cs polymorphism and-optionally one or more otheralleles of other genes or markers close to this gene, which are capableof modifying the risk of developing Alzheimer's disease compared with anormal population.

These vectors may be used for the production of transfected eukaryoticcells or of transgenic animals.

Another subject of the invention therefore consists of the eukaryoticcells transfected by means of a vector as defined above or transgenicanimals produced by means of such a vector.

The results of a study of the levels of expression of the mRNAs for theAPOE gene in the brain will be given below.

EXAMPLE 1

Expression, in the Brain, of the APOE Gene in Patients Suffering fromAlzheimer's Disease Compared with Healthy Controls

1) Extraction of the RNA and amplification of the transcripts

The tissues were chosen in controls aged 65 years or over and inpatients suffering from late onset Alzheimer's disease, whose diagnosishas been confirmed by neuropathological examination, with heterozygousAPOE genotypes.

The extraction of the RNA was carried out on samples of frontal lobes asdescribed in J. Biol. Chem. 247, 4621-4627 (1972), or using anextraction kit (QIAGEN) and then the RNA was digested with DNase(Eurogentec). The RT-PCR was carried out with the aid of the primersdescribed in J. Lipid. res. 31, 545-548 (1990). The reversetranscription reaction was carried out for 1 h 30 min at 37° C., withthe aid of primer F4 (5′-ACAGAATTCGCCCCGGCCTGGTA-3′) at 50 pmolar with 1μg of total RNA as template for the M-MLV reverse transcriptase, inaccordance with the manufacturer's instructions (Gibco/BRL). The PCR wascarried out at 94° C. for 10 minutes, followed by 30 cycles at 58° C.for 1 minute, 72° C. for 1 minute and 94° C. for 1 minute.

The reaction volume of 25 μl for the PCR contained the primer F6 (5′-TAAGCT TGC CAC GGC TGT CCA AGG A-3′) at 50 pmolar, the dNTPs at 0.5 mM,MgCl₂ at 0.1 mM, triton X-100 at 0.1%, glycerol at 15% andTaq-Polymerase at 0.05 units (Eugentec).

The method of calculating the differential expression of the mRNAs isthat described above.

The OD was estimated by the software ®Imagemaster (Pharmacia).

The length of restriction fragments was determined at 91 bp for theallele ε3, 83 bp for the allele ε2 and 72 bp for the allele ε4.

The value of the coefficient A corresponds for the genotypes ε3ε4 andε2ε4 to A=91/72 and A=83/72 respectively. The value of the coefficient Acorresponds for the genotypes ε2ε3 to A=91/83. The genotype ε2ε3 ischaracterized by the bands at 94 and 83 bp. In this case, given that thetwo alleles ε2 and ε3 give restriction fragments of 91 bp, AODε2=ODε3=OD91 bp.

2) Results

THE RESULTS ARE PRESENTED IN FIGS. 1 TO 4:

FIG. 1 representing the products of amplification by RT-PCR afterstaining;

FIG. 2 representing the expression of the mRNAs of heterozygousindividuals ε2ε3, ε3ε4 and ε2ε4 suffering from Alzheimer's disease andof controls. The black lines represent the mean values. The arrowsrepresent the expected values of the level of expression of the ε4allele in the individuals with the ε2/ε4 genotype. These values wereestimated from the percentages observed for the alleles ε2 and ε4 in theindividuals with the ε2/ε4 and ε3/ε4 genotypes, respectively.

FIG. 3 represents the measurement of the level of expression of theallele ε4 in young healthy subjects presenting no cognitive disorders atthe time of sampling and in demented subjects presenting a probablediagnosis of Alzheimer's disease.

FIG. 4 represents the percentage of expression of the ε4 allele in thepatients and controls as a function of the Th1/E47cs and −491 ATgenotypes. The control individuals are represented by the circles andthe sick individuals by the triangles. The black motifs correspond tothe individuals genotyped heterozygous for the −491 AT polymorphism. Themean values for expression are represented by the black bars.

The number of subjects was 49 for the patients suffering fromAlzheimer's disease and 45 for the controls.

The results show that the expression of the mRNA for the ε3 allele was,in all cases, higher than that of the mRNA for the ε4 allele, this inall the individual cases. This result is consistent with the knownmeasurements of the levels of the APOE proteins found in the brains ofindividuals whose genotype has been determined. In this study, it hasbeen shown that the level of APOE in the ε3 homozygotes was higher thanthat for the ε3ε4 heterozygotes which was itself higher than that forthe ε4 homozygotes. Added to each other, the results may suggest eitherthat there is a difference in stability of the different mRNA species orthat there is a genetic variability in the expression of the two allelesin disequilibrium with the genetic polymorphism. However, given that aclear and consistent difference further exists in the ratio of allelicexpression between the patients and the controls, the second hypothesisis probably the correct one. The patients suffering from Alzheimer'sdisease present a higher relative expression of ε4 mRNA than thecontrols (34.9±2.7% against 22.9±2.4 respectively; p<0.0001 by a Mannand Whitney nonparametric test) (FIG. 1). An increase in expression ofthe ε4 allele is also detected in the patients with the ε2ε4 genotypecompared with the controls of the same genotype (46.0±5.4% against28.0±2.8%, p<0.01). On the other hand, a significant decrease in therelative expression of the ε2 allele is demonstrated in the brain of thepatients with the ε2ε3 genotype compared with the controls of the samegenotype (32.8±4.5% against 47.8±3.9%, p<0.002) (FIG. 1). Confirming thedata obtained, the values measured in the sick or control individualswith the ε2ε4 genotype correspond to the values estimated from the othergenotypes (FIG. 1).

With the aim of knowing if the difference of expression of the allelesof APOE is specific to the brain or can be found in other tissues whichare easier to obtain from the living patient (lymphocytes, fibroblastsand the like), the authors have studied the diffential expression of thealleles of APOE in the lymphocytes. The results relating to this studywill be given below.

EXAMPLE 2

Expression, in the Lymphocytes, of the APOE Gene in IndividualsSuffering from Alzheimer's Disease and Young Individuals Presenting, atthe Time of the Study, no Cognitive Disorders

1) Separation of the lymphocytes and culture

A blood sample is collected from individuals probably having Alzheimer'sdisease and from young individuals having no cognitive disorders at thetime of the study. All these individuals have the specific feature ofhaving the ε3ε4 genotype.

The separation of the lymphocytes was carried out on Leucosep tubecontaining a Ficoll gradient. After several washings of the lymphocyteswith RPMI, they are resuspended in a culture medium composed of RPMI,10% FCS, 2 mM glutamate, 100 μM streptopenicillin and 1% ofphytohaemaglutinin (Gibco/Brl). The cell concentration is then 1 millioncells per ml. The cells in suspension are cultured for 48 hours forexample.

2) Extraction of the RNA and amplification of the transcripts

The methods used are those described above in Example 1.

3) Results

The authors observe a higher level of expression of the ε3 allelecompared with the ε4 allele in all the individuals studied (whether theyare sick or controls), thus corresponding to the observations made inthe cerebral tissue. In the lymphocytes, the patients express the ε4allele at a level close to that observed at the level of the brains ofpatients with the ε3ε4 genotype. Out of the 7 potentially sickindividuals, all exhibit a level of expression of the ε4 allele similarto that obtained from cerebral samples from individuals for whom thediagnosis of Alzheimer's disease is certain.

The authors studied the relative expression of the ε4 allele in 5individuals with the ε3ε4 genotype and aged 23 to 55 years and having nocognitive disorders at the time of sampling. This is intended todetermine if a modification of the level of expression of the ε4 allelecan be visible in individuals presenting no clinical sign of Alzheimer'sdisease. There could therefore be a possibility of determining early theindividuals at greater risk of developing Alzheimer's disease. Of the 5individuals studied, all exhibited a level of expression close to thatobserved in the brain of the controls. These results suggest that thesensitivity and the specificity of this test is therefore excellent.

In conclusion, it is advantageous to note that the relative level ofexpression of the ε4 allele could be similar in the cerebral tissue ofdefinite Alzheimer-type patients and the lymphocytes of probableAlzheimer-type patients. Likewise, this level of expression could besimilar in these two cell types for people presenting no cognitivedisorders. Up until now, all the hypotheses seeking to implicate therole of APOE in Alzheimer's disease were based on the fact that thedifferent alleles of APOE were expressed in an equivalent manner and atidentical concentrations in the brain. The results of the presentinvention show for the first time that this is not the case and that thealleles are expressed differently and that this difference issignificantly more marked in patients suffering from Alzheimer's diseasecompared with the control population. The same type of study for theother heterozygous genotypes of the APOE gene as well as an absolutequantification of the APOE gene regardless of the genotype is inprogress.

In conclusion, the results obtained up until now are on agreement withthe existence of a mutation in the regulatory regions of the APOE gene.

The results which led to the demonstration of the Th1/E47cs polymorphismwill be given below.

EXAMPLE 3

Characterization of the Th1/E47cs Polymorphism and its Impact inAlzheimer's Disease.

1. Sequencing of APOE

A fragment of 375 base pairs was amplified by PCR with the primers forthe sense strand 5′TACTTTCTTTCTGGGATCCAGG 3′ and for the antisensestrand 5′ACTCAAGGATCCCAGACTTG 3′. The amplification is carried out over35 cycles (temperature for hybridization of the oligonucleotides: 53°C.) in a buffer containing 1 mM MgCl₂ final. The fragment obtained wassequenced according to the conditions described in the kits forpretreatment (US 70993-Amersham) and sequenceing (USB-Amersham-T7 PCRproduct sequencing kit).

2. Detection of the polymorphism poly LBP1 by restriction enzyme

The amplification of a fragment of 228 base pairs containing the polyLBP1 polymorphism was carried out over 40 cycles in the presence of 0.5mM MgCl₂, 0.5% DMSO. The temperature for hybridization of theoligonucleotides is 53° C. The antisense oligonucleotide is identical tothat used for the sequencing. The sense oligonucleotide is thefollowing: 5′AGAATGGAGGAGGGTGCCTG 3′. The modified nucleotide permittingthe creation of a cleavage site with the G allele for the restrictionenzyme Bstn I is represented in bold. The digestion is carried out at60° C. overnight. The products of digestion are separated on a 12%polyacrylamide gel (acrylamide:bisacrylamide 19:1) and stained in anethidium bromide bath. The T allele is characterized by a restrictionfragment at 49 bp and the G allele by a restriction fragment at 31 bp.

3. Detection of the polymorphism by annealing of oligonucleotidesspecific for each allele (Annealing Specific Oligonucleotide or ASO)

The ASO technique used to detect the poly LBP1 polymorphism is based onthe protocol described by Tiret et al., (1994, Lancet, vol. 344,910-913). The modifications specific to the detection of the poly LBP1polymorphism relating to the oligonucleotides and the washingtemperatures are the following: oligonucleotide specific for the Gallele has the sequence 5′GCCCAGTAATCCAGACACCC 3′ with a washingtemperature 61° C., the oligonucleotide specific for the T allele hasthe sequence: 5′GCCCAGTAATACAGACACCC 3′ with the washing temperature 62°C.

4. Results

The first European population used in this study comprises 310 controls(age=73.5±10.9; 37.3% men) and 293 subjects suffering from early andlate sporadic forms of Alzheimer's disease (age=74.6±9.3; age at thestart of the disease=71.0±8.9; 32.2% men). Three polymorphisms of thelocus of APOE were tested on this population: the Th1/E47cs polymorphismusing the methods described above, the 1E1 polymorphism according to thetechnique used by Mui et al., (Neurology, 1996, vol. 47, 196-201), thepolymorphism of APOE according to the technique of Hixon and Vernier (J.Lipid. Res. vol. 31, 545-548). The number of subjects varies accordingto the polymorphism studied: study of Th1/E47cs, 279 sick individualsand 310 controls; study of 1E1, 293 sick individuals and 307 controls.

In order to improve the statistical power of the study for the analysesat the level of the individuals heterozygous for the APOE genotype, theauthors increased the number of individuals of ε3/ε4 genotype-by addingsubjects of this genotype from independent populations so as to finallyobtain a total of 152 sick individuals and 91 controls (respectively73.3±8.5 and 70.3±8.5 years).

The distribution of the three polymorphisms studied is presented inTable 1. No deviation relative to the Hardy-Weinberg equilibrium wasobserved for each of these polymorphisms.

The tendency to develop the disease is expressed by a factor forapproximating the relative risk OR (for “Odd Ratio”).

CI represents the confidence interval at 95%.

The results are presented in Tables 1 to 5.

As expected, the ε4 allele is closely associated with Alzheimer'sdisease (OR=4.67 CI 95% [3.28-6.67]), whereas the ε2 allele shows aprotective effect (OR=0.27 CI 95% [0.14-0.52]). In the controlpopulation, the G allele of Th1/E47cs is the most represented (0.531)contrary to what is observed in the sick population (0.468). The allelicand genotype distributions are significantly different in the sickindividuals compared with the controls (respectively p=0.03 andp=0.007). The T allele of Th1/E47cs is associated with an increased riskof developing Alzheimer's disease (OR=1.29 CI 95% [1.02-1.63]) and theOR is 1.79 (CI 95% [1.21-12.65], p=0.002) for the individuals carryingat least one T allele. For the 1E1 polymorphism, the allelic andgenotype distributions are significantly different between the twopopulations (respectively p=0.002 and p=0.0005). The C allele of 1E1 isassociated with a decrease in the risk of developing the pathology(OR=0.56 CI 95% [0.40-0.78], p=0.0003).

The authors calculated the linkage disequilibrium, in pairs, for thedifferent polymorphisms studied (Table 2). A significant linkagedisequilibrium exists between the Th1/E47cs, 1E1 and APOE polymorphisms.The authors then estimated the haplotypes generated by these threepolymorphisms using the Myriad Haplotype algorithm (Table 3). Theestimated distribution of the haplotypes is significantly differentbetween the population of sick individuals and that of the controls.Moreover, in order to take into account the strong association of the ε4allele with Alzheimer's disease, the authors compared the estimateddistribution of the haplotypes in the subgroups of ε3/ε3 and ε3/ε4genotype, this distributions being significantly different between thepatients and the controls (Table 3). These results suggest that theTh1/E47cs and 1E1 polymorphisms have a specific effect compared with theε polymorphisms of APOE.

In the general population (patients=261 and controls=253), the authorsestimated the risk of developing Alzheimer's disease for individualspossessing at least one allele of each of the polymorphisms, thisestimation being made in the first instance independently between thesepolymorphisms. A significant effect is then observed for thepolymorphisms (Table 4). When all the polymorphisms are taken intoaccount simultaneously in a logistic regression, the effects of Th1/E47and 1E1 persist and even increase, supporting the hypothesis thatseveral polymorphisms might exist inside the locus of APOE.

If the level of expression of the alleles of APOE is increased by theexistence of mutations in cis in the promoter region of the APOE gene,three implications should be verified: (1) the mutation situated in thepromoter should modulate the expression of the alleles of APOE, thusrevealing the deleterious effect of the ε4 allele or accentuating theprotective effect of the ε2 allele; (2) given that what determines therisk would be the relative level of expression of the two alleles ofAPOE in the heterozygous individuals, this mutation should not have thesame impact in the population of genotype heterozygous for APOE comparedwith the population of homozygous genotype. (3) the effect of a mutationin cis in the heterozygous individuals should depend on the phase ofthis polymorphism with the alleles of APOE, (i.e. the haplotype). In theindividuals carrying a copy of the ε4 allele heterozygous for thegenotype of Th1/E47cs, the ε4 allele may be associated either with the Tallele, or with the G allele of Th1/E47cs. One of these combinationsought to increase the relative level of expression of the ε4 allele andto decrease that of the ε3 allele, the other combination then having theopposite effect.

Thus, in the individuals of ε3/ε4 genotype, the risk of developingAlzheimer's disease will be different between the individualsheterozygous and homozygous for Th1/E47cs. To test these hypotheses, theauthors studied the risk of developing the pathology (adjusted on thesex and the age) of the individuals heterozygous for Th1/E47cs comparedwith the individuals homozygous for this same polymorphism. Only theindividuals heterozygous for the Th1/E47cs genotype and for that of APOEexhibit an increase in the risk of developing the pathology (OR=2.40 CI95% [1.40-4.12], p=0.001), this effect not being found for theindividuals homozygous for the APOE genotype (OR=1.02 CI 95%[0.67-1.57], p=0.91). This impact is demonstrated in particular in thesubpopulation of ε3/ε4 genotype since the risk of developing Alzheimer'sdisease for the individuals heterozygous for the Th1/E47cs genotype isincreased compared with the individuals homozygoug for this genotype(OR=1.90 CI 95% [0.97-3.70], p=0.06; patients=107 and controls=54). Thisincrease in the risk becomes significant by extending the population ofε3/ε4 genotype (OR=2.07 CI 95% [1.21-3.54], p=0.008; patients=152 andcontrols=91) (Table 5). By using a step by step logistic regression, theauthors have shown that the risk of developing the pathology is modifiedonly in the individuals heterozygous for the Th1/E47cs genotype(OR=1.89, εI 95% [1.07-3.35], p=0.027) and not in the individualsheterozygous for the 1E1 genotype. These results therefore suggest thatbetween the two polymorphisms Th1/E47cs and 1E1, Th1/E47cs would be abetter candidate for modifying the relative level of expression of thealleles of APOE.

In order to apprehend the influence of the phase of the Th1/E47cs andAPCE polymorphisms on the risk of developing Alzheimer's disease, theauthors estimated the frequencies of the haplotypes corresponding toassociation of the T or G alleles of Th1/E47cs with the ε4 allele in theindividuals of the ε3/ε4 genotype either for the patients or thecontrols. For the individuals carrying the ε3/ε4 and GT genotypes, 57.6%of the controls are thought to exhibit the association of the T and ε4alleles on the same chromosome, this proportion passing to 69.7% in thepatients. The estimated OR is then 1.7 for the individuals exhibitingthis haplotype, a result which again suggests that the Th1/E47cspolymorphism is capable of influencing the level of expression of the ε4allele.

To confirm these results, the authors studied the impact of theTh1/E47cs polymorphism in a larger population of cases and controls.Furthermore, they combined with this study a new polymorphism in thepromoter of the gene for APOE, −491 AT (Bullido et al., Nat. Genet,1998), which is also capable of modifying the level of expression ofAPOE. The selected population comprises 573 sporadic cases probablyhaving Alzheimer's disease (age=73.8±8.1 years, age at the start of thedisease=70.4±7.9 years, 35.9% men) and 509 controls (age=74.9±9.9 years,35.9% men).

As above, this population is adapted in order to study the impact of theAPOE gene in Alzheimer's disease, since the ε4 allele is stronglyassociated with the pathology (OR=5.40 CI 95% [4.11-7.09], p<0.0001)whereas the ε2 allele exhibits a protective effect (OR=0.47 CI 95%[0.31-0.70], p=0.0003). The frequency of the T allele of the Th1/E47cspolymorphism in increased in the cases compared with the controls (Table6) and the risk of developing AD for the individuals carrying at leastone T allele is 2.13 (CI 95% [1.61-2.83]).

At the level of the study of the −491 AT polymorphism, first of all theauthors observed a similar distribution of the allelic and genotypefrequencies to those described above in the North American population.The frequency of the T allele of the −491 AT polymorphism is reduced inthe patients compared with that of the controls and the OR associatedwith the risk of developing AD for the individuals carrying at least oneT allele of the −491 AT polymorphism is 0.67 (CI 95% [0.52-0.88],p=0.004) (Table 4).

In order to eliminate a possible bias due to the ε4 allele on theassociation of the two Th1/E47cs and −491 AT polymorphisms with AD, theauthors tested the effects of each allele using a logistic regressionadjusted on the presence or the absence of the ε4 allele. Afteradjustment, the risk associated with the presence of at least one Tallele of the Th1/E47cs polymorphism persists (OR=1.56 CI 95%[1.15-2.11], p=0.004), whereas the risk associated with the presence ofat least one T allele of the −491 AT polymorphism disappears (OR=0.82 CI95% [0.62-1.10], p=0.19). This observation suggests that the effect ofthe T allele of Th1/E47cs is not explained by the presence of the ε4allele. As suggested above during the first study, if it is assumed thatthe level of expression of the alleles of APOE is increased or decreasedas a function of cis mutations in the region of the promoter, thesemutations should have a different and detectable effect mainly in theε2/ε3/ε4 heterozygous individuals. In order to verify this hypothesis,the ORs associated with the risk of developing AD were studied in theindividuals heterozygous and homozygous for the APOE genotype using alogistic regression adjusted on age, sex and the presence of at leastone T allele of the Th1/E47cs and −491 AT polymorphisms. In the ε2/ε3/ε4homozygous individuals, no effect is detected (OR=1.13, CI 95%[0.77-1.65] and OR=0.99 CI 95% [0.68-1.44] for the individuals carryingat least one T allele of Th1/E47cs and −491 AT, respectively). On theother hand, in the ε2/ε2/ε4 heterozygous individuals, those who possessat least one T allele of Th1/E47cs exhibit an increase in the risk ofdeveloping the pathology (OR=3.57 CI 95% [2.22-5.75], p<0.0001),contrary to those carrying at least one T allele of −491 AT who exhibita protective effect (OR=0.57 CI 95% [0.37-0.90], p=0.014]). Theseobservations are therefore in agreement with the authors' initialhypotheses.

Thus, the authors' results, obtained by two epidemiological studies,show that the promoter region of the APOE gene possess mutations capableof promoting the development of Alzheimer's disease, in particular theTh1/E47cs polymorphism, independently of the polymorphisms of the APOEgene. This polymorphism modifies the impact of the ε4 allele in thepopulation studied, defining within the ε3/ε4 population, subpopulationspresenting different risks, the individuals being heterozygous for theTh1/E47cs polymorphism presenting the highest risk. The estimation ofthe phase makes it possible to better understand the subpopulation mostat risk, that is to say that exhibiting the combination of the ε4 alleleand of the T allele on the same chromosome.

The influence of these polymorphisms can be explained by an increase inthe level of expression of the ε4 allele or a decrease in the expressionof the ε2 allele relative to the other alleles, this hypothesis beingsupported by the analysis of the differential expression of the mRNAsderived from the alleles of APOE in the brain of the patients and of thecontrols.

It will be important to study the phase between these two polymorphismsusing alleles specific for each of the polymorphisms.

Thus, in a first instance, by two epidemiological studies, the authorshave demonstrated the specific effects of two polymorphisms on the riskof developing AD, the T allele of Th1/E47cs and of −491 AT havingrespectively a deleterious and protective effect. In a second instance,the authors observed a marked differential expression of the alleles ofAPOE, the ε4 allele being overexpressed in the patients of ε3ε4 and ε2ε4genotype, and the ε2 allele being underexpressed in the ε2ε3 patientscompared with the controls of the same genotype. Finally, in agreementwith the data deduced from the studies of the control cases, the levelof expression of the ε4 allele in the brain of the ε3ε4 patients iscorrelated with the mutations present in the regulatory region of theAPOE gene. The T allele of Th1/E47cs increases the relative level ofexpression of the ε4 allele in the patients, whereas the T allele of−491 AT decreases it (FIG. 3 and Table 5). These results are inagreement with recent data, suggesting a modification of the expressionof the APOE gene by these two mutations in a hepatoma cell line.

It is important to note that the Th1/E47cs polymorphism is localized ina consensus site for binding of the transcription factor Th1/E47 andmore particularly in the site for binding of the helix-loop-helixtranscription factor E47. This factor belongs to the class A proteinswhich are expressed ubiquitously and form multiple combinations with theclass B proteins for controlling the tissue-specific expression of genes(Murre et al., Cell. Vol. 15, 451-459). In particular, E47 is expressedin the brain, combined with multiple class B proteins and involved inthe development and maintenance of the nervous system in mammals. On theother hand, little is known about the transcription factor Th1,identified during a screening of mouse embryo cDNA library. A homologousprotein had been described in drosophila, combined with the class Atranscription factor, Daughterless, whose expression is essential inneurogenesis.

In conclusion, all the data gathered by the authors suggest that thestudy of the expression of the APOE gene would make it possible to carryout a diagnosis in individuals heterozygous for the APOE genotype, butalso to better define subpopulations at risk. This information willtherefore be important for defining new therapeutic targets and forprescribing the optimum treatments.

TABLE 1 Combination of the different alleles of the APOE Th1/E47cs and1E1 polymorphisms in the control and patient populations. Allelicfrequency Genotypic frequency APOE N ε2 ε3 ε4 ε2/ε2 ε2/ε3 ε2/ε4 ε3/ε3ε3/ε4 ε4/ε4 Controls 308 0.081 0.805 0.114 0.003 0.133 0.230 0.649 0.1790.130 Patients 292 0.034 0.639 0.327 — 0.034 0.034 0.425 0.393 0.113Th1/E47cs N G^(a) T GG^(b) GT TT Controls 310 0.531 0.469 0.320 0.4220.258 Patients 279 0.468 0.532 0.208 0.520 0.272 1E1 n G^(c) C GG^(d) GCCC Controls 307 0.615 0.385 0.388 0.456 0.156 Patients 293 0.711 0.2890.533 0.382 0.085 N ε2/ε3* ε3/ε3 ε3/ε4 ε4/ε4 ε2/ε4 Combination of theAPOE and 1E1 polymorphisms Patients CC 22 1 20 — 1 — GC 100 4 50 46 — —GG 139 4 44 56 27 8 n 261 9 114 102 28 8 Controls CC 41 0 41 0 — — GC112 12 76 24 — — GG 100 25 50 15 3 7 n 253 37 167 39 3 7 Combination ofAPOE and Th1/E47cs polymorphisms Patients GG 55 4 36 13 1 1 GT 134 5 5460 8 7 TT 78 — 24 29 19 — n 261 9 114 102 28 8 Controls GG 85 25 52 5 —3 GT 108 11 76 16 1 4 TT 60 1 39 18 2 — n 253 36 167 39 3 7 ^(a)p <0.001: ^(b)p = 0.03. OR (T**/T*) = 1.29 CI 95% [1.02-1.63]: ^(c)p =0.007: ^(d)p = 0.002. OR (G**/G*) = 1.44 CI 95% [1.14-1.84]: ^(e)p =0.0005: ^(f)NS: one individual is genotyped ε2/ε2 in the controlpopulation.

TABLE 2 Disequilibrium of the linkage in the chromosomal region 19q13.2region. Th1/E47cs 1E1 APOE Cases Th1/E4 7cs — −8.4 −5.8 1E1 79 — −6.1APOE 49 54 — Controls Th1/E47cs — 11.9 −1.9 1E1 86 — −3.7 APOE 32 42 —

The APOE polymorphism was analysed as a biallelic marker (i.e. allele 4versus allele 2 or 3)

The standardized coefficient Δ of linkage disequilibrium is representedabove the diagonal of the table. The maximum percentage D′ of thelinkage disequilibibrium for the given allelic frequencies isrepresented below this diagonal.

TABLE 3 Estimation of the possible haplotypes between the differentpolymorphisms studied in the chromosomal region 19q.13.2. The anonymousmarker D19S178 was integrated into this study. Estimated frequencies ofhaplotypes Haplotype Polymorphisms Controls Number D19S178 Th1/E47cs 1E1APOE Cases Controls expected a. General population 1 S T G ε4⁻ 0.1300.032 0.029  2 S T C ε4⁻ 0.004 0.000 0.019* 3 S G G ε4⁻ 0.031 0.0060.035* 4 L T G ε4⁻ 0.100 0.043 0.027  5 L T C ε4⁻ 0.005 0.000 0.018* 6 LG G ε4⁻ 0.044 0.022 0.033  7 L G C ε4⁻ 0.003 0.000 0.021* 8 S T G ε4⁻0.018 0.009 0.114* 9 S T C ε4⁻ 0.127 0.183 0.072* 10  S G G ε4⁻ 0.1840.267 0.142* 11  S G C ε4⁻ 0.000 0.011 0.087* 12  L T G ε4⁻ 0.020 0.0140.113* 13  L T C ε4⁻ 0.128 0.170 0.070* 14  L G G ε4⁻ 0.195 0.224 0.136*15  L G C ε4⁻ 0.011 0.019 0.084* Number of chromosomes ε4⁻ 522 506 ε4⁻b. Population ε3/E3 genotype 1 S T G 0.035 0.014 0.129* 2 S T C 0.1920.239 0.116* 3 S G G 0.259 0.268 0.151* 4 S G C 0.009 0.013 0.136* 5 L TG 0.027 0.010 0.113* 6 L T C 0.193 0.198 0.103* 7 L G G 0.285 0.2340.133* 8 L G C 0.000 0.024 0.119* Number of chromosomes 228 334 c.Population ε3/ε4 genotype 1 S T G 0.184 0.154 0.207  2 S T C 0.102 0.1480.092  3 S G G 0.169 0.145 0.103  4 s G C 0.000 0.000 0.046  5 L T G0.179 0.204 0.254  6 L T C 0.113 0.158 0.127  7 L G G 0.237 0.188 0.113 8 L G C 0.015 0.000 0.056  Number of chromosomes 204  78

TABLE 4 ORs estimated by multiple logistic regression Th1/E47cs 1E1 APOEwith OR 95% CI TT + GT/GG CC + GC/GG ε4/without ε4 Non adjusted 1.79[1.21-2.65] 0.56 [0.40-0.78] 4.32 [2.98-6.28] P = 0.002 p = 0.0003 p <0.0001 Adjusted on 1.90 [1.28-2.83] 0.58 [0.41-0.82] 4.66 [3.14-6.93]the sex and age P = 0.002 p = 0.002 p < 0.0001 Adjusted on the 2.51[1.38-4.56] 0.43 [0.25-0.74] 3.22 [2.06-5.02] sex, age and P = 0.001 p =0.002 p < 0.0001 other polymorphisms

TABLE 5 Distributions of the 1E1 and Th1/E47cs genotypes in the extendedε3ε4 population. 1E1 polymorphism^(a) Th1/E47cs polymorphism^(b) N GG GCGG GT TT Controls 91 50 41 16 38 37 Cases 152 67 85 19 89 44 ^(a) =0.10; ^(b) = 0.04.

TABLE 6 Allelic and genotypic distribution of the APOE Th1/E47cs and-491 AT polymorphisms. Allele Genotype APOE ε2 ε3 ε4 ε2ε2* ε2ε3 ε2ε4ε3ε3 ε3ε4 ε4ε4 Controls   75(0. 832(0 111(0    4(0.0   57(0.   10(0.344(0   88(0.  6(0.0 Cases   40(0. 699(0 407(0 —   16(0.   24(0. 223(0237(0 73(0. Th1/E47cs G T GG* GT TT Controls 562(0 456(0 162(0 238(0109(0 Cases 515(0 631(0 103(0 308(0 162(0 -491 AT A T AA^(I) AT. TTControls 833(0 185(0 343(0 147(0   19(0. Cases 993(0 153(0 432(0 129(0  12(0. The number of alleles (frequency) is presented. The genotypicdistributions are in Hardy-Weinberg equilibrium in the controlpopulations. *p < 10⁻⁴; ^(I)p = 0.005.

TABLE 7 Relative expression of the ε4 allele in the patients and thecontrols as a function of the Th1/E47cs and -491 AT polymorphisms. nCases n Controls* Th1/E47cs GG 4 33.5 ± 1.7% 4 22.5 ± 2.5% GT 19 36.3 ±2.1% 11 22.6 ± 1.9% TT 10 32.4 ± 1.4% 10 22.8 ± 2.7% -491 AT AA 26 35.3± 2.7% 15 22.8 ± 2.2% AT 7  33.5 ± 1.3%^(§) 10 22.5 ± 2.3% *NS. : P<10⁻³, ^(§)P = 0.11.

What is claimed is:
 1. A method of detecting the susceptibility of apatient to Alzheimer's disease, which comprises (a₁) determining thelevel of expression of the ε4 allele of the gene encoding apolipoproteinE in patients heterozygous, ε4/ε3 or ε4/ε2, for the apolipoprotein Egenotype ε, wherein susceptibility to Alzheimer's disease is indicatedby the presence of a mutation in the consensus sequence binding theTh1/E47cs transcription factor and an increase in the level ofexpression of the ε4 allele compared to a control population; or (a₂)determining the level of expression of the ε2 allele of the geneencoding apolipoprotein E in patients carrying the apolipoprotein Egenotype ε2/ε3, wherein susceptibility to Alzheimer's disease isindicated by the presence of a mutation in the consensus sequencebinding the Th1/E47cs transcription factor and a decrease in the levelof expression of the ε2 allele compared to a control population.
 2. Themethod according to claim 1, further comprising (b) identifying a G to Tsubstitution within the consensus binding sequence TH1/E47cs at 186bases from the first nucleotide of the TATA box of the gene encodinghuman apoE, wherein the presence of the mutation indicates asusceptibility to Alzheimer's disease.
 3. The method according to claim1, wherein a biological sample selected from the group consisting ofcerebral tissue, lymphocytes, fibroblasts and other tissues capable ofexhibiting a difference in expression of ε alleles of the apolipoproteinE gene is used to identify the mutation and to determine the levels ofexpression of the ε4 or ε2 allele with respect to the other allele.
 4. Amethod of detecting the susceptibility of a patient to Alzheimer'sdisease independent of an ε polymorphism, which comprises identifying amutation in the promoter region of the gene encoding apolipoprotein E inthe consensus sequence binding the Th1/E47cs transcription factors;wherein the presence of the heterozygous mutation indicates asusceptibility to Alzheimer's disease.
 5. The method according to claim4, wherein the mutation exists in a region situated at 186 bases fromthe the first nucleotide TATA box of the gene encoding human apoe. 6.The method according to claim 4, wherein a biological sample selectedfrom the group consisting of cerebral tissue, lymphocytes, fibroblastsand other tissues capable of exhibiting a difference in expression of εalleles of the apolipoprotein E gene is used to identify the mutation.